The invention relates to a flow cytometer which radiates a laser beam toward a flow of aqueous solution containing cells, chromosomes, and biopolymers included therein, thereby detecting fluorescence and scattered light radiated from the cells or the like, to thus classify and count the number of the same.
Flow cytometers have been in wide use: (1) for measuring relative amounts, between cells, of DNA, RNA, enzyme, protein, and the like; (2) for examining functions, such as cell activity, antibody productivity, and enzyme activity; and (3) for automatically classifying types of cells, chromosomes, lymphocytes, or the like.
A conventional example of a flow cytometer of this type has the following configuration. A sample solution is prepared by, for instance, diluting and staining a blood sample. The sample solution is supplied in a form of a narrow flow to a center section of a flow cell. The narrow flow is irradiated with narrowly-converged light emitted from a light source, thereby forming a detection section. Every time a blood cell passes through the detection section, changes in scattered light and fluorescence are detected by a photodetector. A two-dimensional scattergram, wherein a scattered light intensity and a fluorescence intensity form two axes, is produced on the basis of the thus-detected signals. Demarcation lines are set on the two-dimensional scattergram, whereupon classification and counting of the particles are performed.
FIG. 3 shows such a flow cytometer comprising a laser light source 10b which is used for exciting both of scattered light and fluorescence. A laser beam emitted from the laser light source 10b is radiated at a target particle to be measured (hereinafter referred to as a target particle) 15 in a flow cell 14 by way of a beam shaping lens 12. As described above, the laser beam radiated at the target particle 15 excites forward scattered light, orthogonal scattered light, and fluorescence, respectively. Meanwhile, the flow cytometer is configured sum that a direct beam of the laser beam radiated toward the flow cell 14 is blocked by a shade 16 disposed in front of the flow cell 14.
As described above, forward scattered light resulting from excitation of the target particle 15 is condensed by a condenser lens 18a, input to a forward scattered light detector 20a, and converted into an electric signal. Meanwhile, the orthogonal scattered light and fluorescence having been excited on the target particle 15 are condensed by a condenser lens 19a, and brought incident to serially-arranged beam splitters 22a, 22b, 22c, and 22d in sequence.
The orthogonal scattered light is reflected by the beam splitter 22a. The thus-reflected optical beam is condensed by a lens 18b, input to an orthogonal scattered light detector 20b, and converted into an electric signal. The optical beam transmitted through the beam splitter 22a is subjected to elimination of scattered light wavelength originating from the light source for scattered light excitation or the same originating from the light source for exciting fluorescence by a wavelength filter 33, and thereafter caused to enter the serially-arranged beam splitters 22b, 22c, and 22d in sequence. A first fluorescence having been set in advance is reflected by the beam splitter 22b. The reflected light beam is condensed by a condenser lens 19b by way of a wavelength filter 24a, input to a fluorescence detector 26a, and converted into an electrical signal. Similarly, a second fluorescence having been set in advance is reflected by the beam splitter 22c. The reflected light beam is condensed by a condenser lens 19c by way of a wavelength filter 24b, input to a fluorescence detector 26b, and converted into an electrical signal. In addition, a third fluorescence having been set in advance is reflected by the beam splitter 22d. The reflected light beam is condensed by a condenser lens 19d by way of a wavelength filter 24c, input to a fluorescence detector 26c, and converted into an electrical signal. Meanwhile, a transmitted light beam is constituted of a fourth fluorescence having been transmitted through the beam splitter 22d. The transmitted light beam is condensed by a condenser lens 19e by way of a wavelength filter 24d, input to a fluorescence detector 26d, and converted into an electrical signal.
According to the flow cytometer of the above configuration, target particles are classified into the respective types of fluorescence as described above, and subjected to measurement; whereby the specific properties of the variety of target particles can be analyzed.
In such a flow cytometer, however, an argon laser is used as a light source for exciting fluorescence of a target particle. The argon laser is used because light of relatively shorter wavelength and in a blue-light region must be used as an excitation light for causing the target particle to emit fluorescence. However, an argon laser is expensive. In addition, not only is the footprint of the laser apparatus per se large, but also accompanying peripheral devices, such as a required power source for driving the laser, and the like, are also large. Furthermore, overall power consumption by the argon laser is also large.
In addition, when target particles differ, types of fluorescence to be analyzed differ. Accordingly, setting of sensitivities and threshold values of the respective detectors must be optimized by, for instance, changing settings of the wavelength filters. The operations to perform such changing are considerably complicated. Therefore, for conducting analysis of a variety of target particles, the following method would be easy and convenient. That is, target particles are specified in advance; and, on the basis thereof, a plurality of flow cytometers, in which sensitivities and threshold values for the respective wavelength filters are optimized, are installed. However, of a flow cytometer, a light source and peripheral equipment thereof are expensive. Accordingly, installation of a flow cytometer for each type of the target particles involves enormous installation cost and an enormous footprint, which is economically disadvantageous.